Local anesthetics (LAs) interact intimately with membranes in order to reach their pharmacological targets. Blockade of sodium channels and of other neuronal proteins appears to occur at sites that must be reached by drug molecules that have penetrated into and/or through the plasma membrane. In addition, for peripheral nerve block, LAs must penetrate the ensheathing membranes composing the peripheral nerve's perineurium, before they reach the actual nerve cells. Therefore, both the efficacy and safety of LAs depend on their membrane-penetrating abilities. In this application we address the long-term objectives of identifying the various factors that determine LA: membrane interactions and thereby understanding the mechanism of drug permeation through bilayer membranes. Five Specific Aims frame the experimental approach: 1. Measurements of equilibrium membrane partitioning and of membrane permeability of LAs; 2. Measurements of intramolecular bonding of LAs in solvents that model membrane regions; 3. Measurements of membrane "fluidity" and of its modification by LAs. 4. Correlate the the measures in Aims 1-3 in order to determine the relative contributions of LA hydrophobicity, charge, dipole moment and intramolecular bonding to membrane partitioning and to permeation, and the relative importance of the membrane fluidity, surface charge, presence of cholesterol, dipolar field potential and biological (tissue) source, for LA:membrane interactions. Both the structure of the LA and the composition of the membranes will be systematically altered to manipulate these various factors. 5. Permeability and equilibrium binding of the LAs used in Aims 1-3 will be measured for mammalian epithelial membranes, where transport more closely resembles the passage of drugs across the barriers ensheathing nerve tissue. Novel spectrofluorometric methods will be applied to determine partition coefficients, the depth of membrane binding of neutral and charged LAs, and the pKa of vesicle membrane-adsorbed drug. A newly developed reporter system, using the fluorescence of a protein that is quenched by ligand binding reactions known to be antagonized by LAs, will be incorporated within vesicles made of the same membranes, for continuous detection of LA influx and calculation of the corresponding permeability. Although the immediate goal of this proposal focuses on understanding LA- membrane interactions, the theory and the methods can be applied to many other drugs that interact with and permeate cellular membranes.